Facscanto ruo orange analyzer

The levels of active caspase-1 were quantified in live cells using FLICA 660 Caspase-1 Assay Kit (ImmunoChemistry Technologies Cat# 9122). This assay employs a fluorescent inhibitor probe 660-YVAD-fmk to label active caspase-1 in living cells. Briefly, following 24h treatment with ssRNA40 or ssRNA41, HMG cells were washed with PBS and incubated with FLICA™ 660-YVAD-fmk (1:60 dilution) for 30min. After two washes with PBS, cells were further stained with Aqua stain (Live/Dead™ Fixable Dead cell Stain Kit; Molecular Probes Cat# L34957). Cells were resuspended in PBS for flow cytometry analysis using BD FACSCanto RUO-ORANGE analyzer. Data were analyzed using FlowJo v10 software (Tree Star). The levels of active caspase-1 were also measured in the culture supernatants using the Human Caspase-1/ICE Quantikine ELISA Kit (R&D Systems Cat# DCA100). This assay specifically measures the active caspase-1 by using an antibody against p20 subunit of caspase-1.

Following incubation with ssRNA40 or ssRNA41 for 24h or 48h, HMG cells were washed with 1X PBS and incubated with MitoSOX Red (Molecular Probes Cat# M36008) for ROS measurement; TMRE (Molecular Probes Cat# T669) or Mitotracker Green and Deep Red (Molecular Probes Cat# M7514, M22426) for measuring mitochondrial membrane potential. Following 10–20 min incubation, HMG cells were washed and collected in PBS for analysis by flow cytometry using BD FACSCanto RUO-ORANGE analyzer. Data were analyzed using FlowJo v10 software (Tree Star).

Flow analysis of freshly isolated monocytes differentiated into MMG was performed after 10–12 days in culture. Fetal microglia cells were isolated from 10-day-old brain cultures and cultured for an additional 2 days. Expression of specific surface molecules and intracellular molecules was studied using flow cytometry. Cells were detached using a scraper and staining was performed according to manufacturer’s protocol. Expression of CD11c, CD11b, CD195, CD14, CD45, HLA-DR, CD80, and CD86 was measured by incubating the MMG and HMG cells with fluorescein isothiocyanate (FITC)-anti-CD11c, AF488-anti CD11b, allophycocyanin (APC)-anti CD80, PerCP-anti-HLA-DR, phycoerythrin-Cy7-anti-CD86 and PerCP-anti-CD14 (all Biolegend), eFluor450-anti-CD45, and APC-anti-CD195 (all eBioscience) on ice for 30 min. Intracellular CD68 was detected using BD Cytofix/CytoPerm kit with PE-anti-CD68 according to manufacturer’s protocol (BD Biosciences). Corresponding labeled isotype control or FMO controls were included in all experiments. For cell viability analysis of HIV-infected MMG, cells were stained with fixable viability dye eFluor506 (eBioscience). The cells were fixed and resuspended in phosphate-buffered saline (PBS) for flow cytometry using BD FACSCanto RUO-ORANGE analyzer. Data were analyzed using FlowJo software.